The Triton X-100 and High Salt Resistant Residue of
Saccharomyces cerevisiae Nuclear Membranes
Karlheinz Mann and D ieter Mecke
Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Straße 1, D-4700 Tübingen
Z. Naturforsch. 37 c, 9 1 6 -9 2 0 (1982); received July 2, 1982
Nuclear Membrane, Triton X-100, High Salt Treatment, Insoluble Membrane Residue
Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting
chromatin with deoxyribonuclease after an initial treatment o f nuclei with very diluted buffers.
When the nuclear membranes were treated with 5% Triton X-100 and 1 m NaCl an insoluble
fibrous net was obtained which consisted mainly o f protein with Mr values of 85000, 48000,
45000, 39000 and 31000. Lamins, a set of proteins with Afr = 6 5 0 0 0 -7 5 0 0 0 , which were shown
to be the major proteins o f the insoluble nuclear membrane residue o f higher eukaryotes, were not
found.
1. Introduction
There are features of Saccharomyces cerevisiae
nuclear structure and behaviour which are rather
different from those o f most higher eukaryotes
[1, 2]. Like in all ascomycetous yeasts and fungi the
nucleus is not broken down during cell divisions.
This kind of nuclear division with intact nuclear
envelope probably dem ands specialized regions of
nuclear membrane, like regions o f m em brane syn­
thesis and assembly, or regions of different m em ­
brane fluidity. O ther specialized regions of the
nuclear envelope are visible with the electron
microscope: the spindle plaques, which serve as
nucleating centres for the m icrotubules of the intra­
nuclear spindle. The pores o f the Saccharomyces
cerevisiae nuclear envelope seem to have a sim pler
structure than those of higher eukaryotes [3] and
they seem to have lateral m obility, leading to
uneven distribution in different stages o f the cell
cycle [3, 4]. It seemed possible to us that these dif­
ferences reflect a m olecular organization o f the
yeast nuclear envelope which could be different
from that of higher organisms with open mitosis.
2. Experimental
nuclear pellet was suspended in 2 m M Tris buffer,
pH 7.5, containing 10% sucrose and 0.05 m M M gCl2.
The suspension was kept in ice for 15 min before
centrifugation at 40000 x ^ max at 2 ° C for 30 min.
The pellet was taken up in 20 m M Tris buffer,
pH 7.5, containing 0.5 m M M gCl2 and 10% sucrose to
obtain a suspension with 2 .5 - 5 mg protein/m l. To
this suspension 400 U of pancreatic ribonuclease
and 4000 U of pancreatic deoxyribonuclease I were
added per 50 mg of nuclear protein. After 90 min
incubation at 2 2 -2 5 °C the m ixture was centri­
fuged as above. The pellet was suspended in the
same buffer and layered onto a discontinuous
sucrose gradient of 35, 42, and 52% sucrose in the
same buffer. After centrifugation at 100000 x g ay at
2 °C for 2 h the nuclear m em brane fraction was
removed from the 42/52% interface and diluted
with 20 m M Tris buffer, pH 7.5, containing 6%
Triton X-100, 1.2 m NaCl and 0.5 m M M gCl2 to a
final concentration of 5% Triton and 1 m NaCl. The
detergent to protein ratio was usually 250-300.
This suspension was gently shaken for 30 min at
2 2 -2 5 °C before centrifugation at 100000 x # av for
30 min at 2 °C . The pellet was suspended in the
usual Tris buffer and centrifuged at 40000 x g miX for
30 min at 2 °C. 1 m M phenylmethylsulfonyl fluoride
was added to all buffers shortly before use.
2.1. Isolation o f nuclear fractions
Nuclei were isolated from the haploid Saccharo­
myces cerevisiae strain SM C-19 A [5] as in [6]. The
Reprint requests to K Mann.
0341-0382/82/1000-0916
$01.30/0
2.2. Analytical methods
Preparation of samples for electron microscopy
was done as in [7]. Gel electrophoresis was done as
in [8] with 5% stacking gel and 12% separating gel.
The Triton/N aC l supernatant was first treated with
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K. Mann and D. Mecke • Nuclear Membranes Pellets o f Saccharomyces
XAD as in [9] and then dialysed against H 20 before
electrophoresis. Lipids and nucleic acids were ex­
tracted as in [6]. Protein was m easured as in [10]
after precipitation as in [11]. Phospholipids, sterols
and neutral glycerides were determ ined as in [12 —
14]. RNA was measured as in [15], and D N A was
estimated by the absorbance of the hydrolysis
products at 260 nm.
3. Results and Discussion
Incubation of nuclei in 2 mM Tris buffer led to
bursting and extrusion o f chrom atin. Lum ps of
chromatin and ribosomes were however still bound
to the membranes. They were rem oved with nu­
cleases. Centrifugation o f the crude m em brane pel­
let in a continuous sucrose gradient from 30-55%
sucrose gave 3 fractions. 4 -8 % o f the total m aterial
applied to the gradient rem ained at the top. It
contained single m em brane vesicles and was very
rich in neutral lipids. A nother 3 -5 % were found in
a pellet containing single m em brane vesicles which
were sometimes filled with residual chrom atin. This
fraction was rich in nucleic acids and neutral lipids.
More than 80% of the total m aterial was found in a
fraction from 42-47% sucrose. The distribution of
material in this fraction was not sym m etrical: ab­
sorption at 600 nm was higher in the denser regions.
Electron microscopic exam ination showed that the
number of large double m em brane vesicles with
many pores increased with denisty. Electrophoresis
showed however exactly the same protein pattern
in different density regions of this fraction. T here­
fore it was collected as a sharp zone at the interface
between 42 and 52% sucrose layers o f a discontinu­
ous gradient for the following experim ents. This
membrane fraction was identified as nuclear m em ­
917
brane fraction because it contained many double
membrane profiles, either as large vesicles or as
sheets, which were studded with pores (Fig. 1). Pores
were much more frequent than in nuclear m em ­
brane fractions treated with high salt buffers [6, 7],
The composition of this fraction is shown in Table I.
The higher recovery of nucleic acids with this isola­
tion method as well as the presence o f some strongly
contrasted grains, possibly ribosomal rem nants, as­
sociated with the membranes (Fig. 1 B) showed that
the better preservation of nuclear envelope struc­
tural elements obtained with this mild procedure
had to be payed with a higher level of im purities,
compared to preparations treated with high salt
solutions [6, 7],
When the nuclear m em brane fraction was ex­
tracted with 5% Triton X-100 in the presence of 1 m
NaCl, an insoluble residue rem ained, which was
composed mainly of protein, but which also con­
tained residual nucleic acids and lipids (Table I). As
in other membranes [16], the neutral lipids were
much more resistant to Triton extraction than phos­
pholipids. Therefore rather high detergent concen­
trations had to be used. Sequential extraction, first
with detergent and then with N aCl, or sim ultaneous
extraction at 0 °C were much less effective. Electron
microscopic examination of the nuclear m em brane
residue showed a fibrous net com parable to re­
sidues isolated from rat liver [17] or am phibian
oocyte [18]. The yeast nuclear m em brane residue
appeared however much denser (Fig. 1 D). Struc­
tural components having the dim ensions of pores
were found less frequently than pores in the unex­
tracted membranes. It is however possible that they
were more difficult to identify than in higher
eukaryote nuclear m em brane residues because of
poor preservation, lack of defined internal struc­
tures, simpler shape, or density o f the surrounding
Table I. Composition and recovery o f nuclear fractions. The compositon is in wt% of the sum o f compounds tested. The
mean values of 3 preparations are shown ± SD. Recovery values are the mean o f 2 preparations.
Protein
Phospholipid
Sterols
Glycerides
RNA
DNA
Nuclei
Nuclear
membranes
Recovery
from nuclei
[%]
Insoluble
residue
Recovery from
membranes
[%]
73.2
6.9
1.8
1.8
14.6
1.9
62.1
20.9
5.7
5.6
4.7
0.7
18.3
71.5
57.0
53.0
7.5
6.8
83.5
1.3
3.5
4.0
4.1
4.1
8.6
0.5
3.5
4.3
5.2
43.0
± 1.9
± 0.6
± 0.2
± 0.3
± 1.2
± 0 .3
±
±
±
±
±
±
2.8
4.5
1.5
2.1
1.7
0.2
± 2.7
± 0.3
± 0.9
± 0.6
± 2.1
± 0 .7
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K. Mann and D. Mecke • Nuclear Membranes Pellets o f Saccharomyces
Fig. 1. (A) Isolated nuclear membranes. 30300 x; (B) large nuclear envelope fragment with double m em brane profile and
pores. 47000 x; (C) nuclear envelope fragment with pores and spindle plaque (arrow), 76000 x; (D ) the Triton X-100 and
NaCl resistant membrane residue, 55 500 x.
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K. Mann and D. Mecke • Nuclear Membranes Pellets of Saccharomyces
1
2
3
4
Fig. 2. Electrophoretic analysis of nuclear membrane frac­
tions. Lane 1, nuclear membrane; lane 2, Triton and high
salt resistant residue; arrows point to major proteins with
Mr 85000, 48000, 45000, 39000, 31000 and 28000; lane 3,
detergent and high salt soluble proteins; lane 4, Mx stan­
dards: a, phosphorylase b (94000); b, bovine albumin
(67000); c, ovalbumin (43000); d, carbonic anhydrase
(30000); e, trypsin inhibitor (20100); f, a-lactalbumin
(14400).
fibrillar net. The polypeptide com position o f the
nuclear m em brane fractions is shown in Fig. 2.
4 major proteins were found in the insoluble residue.
They had M x values of 48000, 45000, 31000 and
919
28000. Other, less prominent, but clearly enriched
proteins had M r values of 85000 and 39000. All of
these proteins, except the 85 K protein, were also
major proteins of the unextracted nuclear m em ­
brane (Fig. 2). Most of them were also present to a
variable extent in the T riton/N aC l soluble fraction.
Such a differential extraction behaviour had also
been found with avian erythrocyte [19] and rat liver
[20] nuclear membrane proteins. The protein pattern
of the yeast nuclear m em brane residue was not so
simple as that of am phibian oocytes, which shows
only 1 major protein with M r = 68000 [21]. The
protein pattern of rat liver nuclear m em brane res­
idue, on the other hand, is dom inated by a triplet of
proteins with M x= 65000-75000 [17]. These pro­
teins, the lamins, were shown to be the m ajor
proteins of the lamina, a protein structure subjacent
to the inner nuclear m em brane in intact envelopes.
During mitosis, when the nuclear envelope of
higher eukaryotes is broken down, the lam ina is
depolymerized [22], Judged from the M r of the
major proteins, lamins, if present at all, seem not to
play a role in the insoluble residue of the yeast
nuclear membrane. At present we do not know
whether other proteins replace them, or w hether the
yeast nuclear envelope has a different structural
organization. Furtherm ore it should be stressed,
that the yeast nuclear membrane residue was iso­
lated from rapidly dividing cells. Therefore it is still
possible that nuclear m em brane residues from rest­
ing yeast cells will show more resemblance to the
pore complex-laminae of higher eukaryotes.
Acknowledgements
This work was supported by the Fonds der
Chemischen Industrie.
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K. Mann and D. Mecke • Nuclear Membranes Pellets of Saccharomyces
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